HISTOPATHOLOGIC TECHNIQUES b.

Requisition form
- Requisition number to identify px
The Histotechnology Process - always include middle initial/name
Assign a number: record keeping; ensure no errors in - sx must include laterality (left/right) esp. if bilateral
identification - source
Prevent autolysis: chemical preservation of the specimen - no RF, no acceptance of sx
Prepare for embedding: tissue dehydration and clearing - info on logbook must tally w/ info on RF
Support tissue for cutting: hardening and supporting the - w/ name of attending physician
material with media (paraffin wax or plastic) c. Tissue preparation
Cutting a very thin slice to let light to pass through to make -size: container
individual components visible ex. small sx = small container
Staining: use of contrasting colors to increase visibility and make multiple sx = separate containers
examination easier *ideal: unbreakable, transparent, w/ cap
Storing the finished product for future reference -fixation: properly preserved
ex. solid = formalin
Tissue Processing slides = 95% ethanol
Biopsies, larger specimens removed at surgery, or tissues from if w/o fixative, note down on RF; not accepted
autopsy taken for diagnosis of disease processes - label: name of px, SP no. (specimen number)
Microscopic slides
- viewed under the microscope by pathologists II. FIXATION
Histotechnologists - preserve tissues permanently in a life-like state
- perform tissue processing; make the glass microscopic slides chemically freezing the tissues permanently in place
- prevention of degeneration, putrefaction, decomposition,
Specimen Accessioning distortion
Request form - lists the patient information and history; protein stabilization (cross links b/w fixative & proteins)
description of the site of origin. - should be carried out ASAP after removal of the tissues or soon
SP number - identify each specimen for each patient after death to prevent autolysis (destruction by enzymes
produced by the tissue itself)
Gross Examination - Reduce risk of infection
*pathologist, pathology assistant, pathology resident - Promotes staining
- examines tissues for diagnosis - Inhibit bacterial decomposition
*describing the specimen placing all or parts of it into a small - Stop all cellular activities
plastic cassette - prevent breakdown of cellular elements
* Cassette Inactivation of lysosomal hydrolytic enzymes post
- holds the tissue while it is being processed to a mortem decomposition (autolysis)
paraffin block by chemically altering, stabilizing, and making tissue
- initially placed into a fixative components insoluble
- dimension: 2 x 1.5 x 0.5 Prevention of putrefaction after death (bacterial/
* Prosector makes the cutting; measures size, weight; color, fungal colonization & overgrowth)
consistency, shape, smell - coagulate or precipitate protoplasmic substances
Additive fixation chemical constituent of fixative is
Inking For Margins taken in & becomes part of the tissue by cross links
- When a malignancy is suspected, the specimen is covered w/
or molecular complexes stable protein (formalin,
ink to mark the margins of the sx
mercury, osmium tetroxide)
- Different colored inks to identify different areas if needed
Non additive fixation removes bound water by
- When sections are made and processed, the ink will mark the
attaching to H bonds of certain groups within the
actual margin on the slide
* India ink black protein molecule new cross links are established
* blue superior margin (alcoholic fixatives)
* green inferior margin
_____________________________________________________ *Accdg to composition:
12 BASIC STEPS/ TECHNIQUES Simple made up of only one subs.
Compound more than one subs
I. NUMBERING
*Accdg to action:
- very important
Microanatomical for ultrastructural components
*3 basic areas:
a. Recording/Logging Cytological nuclear and cytoplasmic
logbook (date, name of px, specimen, sx #) Histochemical preserves chemical components
(ex. mucin, carbs, bilirubin)
*Factors Affecting Fixation
a. Buffering/ Hydrogen Concentration
- Fixation is best carried out close to neutral pH (6-8)
- Hypoxia lowers the pH, so there must be buffering capacity
to prevent excessive acidity
- Acidity favors formation of formalin-heme pigment (black,
polarizable deposits in tissue)
*Common buffers: phosphate, bicarbonate, cacodylate,
veronal
*Commercial formalin - buffered with phosphate (pH 7)
b. Penetration/ Thickness
- depends upon the diffusability of each fixative (constant)
* Formalin & alcohol - best
* glutaraldehyde - worst
* Sectioning thinly (2-3 mm) makes penetration more rapid
*1-2mm/hr
*1 to 2 mm sq. = for EM
*2 cm sq. = light microscopy
*Brain: suspended whole in 10% buffered formalin (2-3 wks)
c. Volume
- 10:1 ratio of fixative to tissue.
* If ratio is less than ideal, change the fixative at intervals to
avoid exhaustion of the fixative.
* Agitation will also enhance fixation
d. Temperature
- room temperature: sufficient to maintain excellent
morphological detail; surgical sx; mast cells
- 0-4 C: For electron microscopy and histochemistry Celcius.
- Nucleic acids do not react with fixatives at room temp.
- Increasing the temperature = increase the speed/rate of
fixation (but can also increase the rate of autolysis)
- Hot formalin - fix tissues faster; often the first step on an
automated tissue processor (45 C)
Formalin at 60 C for very urgent biopsy specimens
Formalin at 100 C used for fixing tissues w/ TB
e. Concentration
- should be adjusted down to the lowest level possible.
* Formalin = 10%
*glutaraldehyde = 0.25% (for immunoelectrochem)- 4%
*Presence of buffer causes polymerization of aldehyde, with
consequent decrease in its effective concentration.
f. Fixation Time
- faster = better
- longer fixation time: more cellular organelles lost; more
nuclear shrinkage & artifactual clumping; may severely
inhibit enzyme activity and immunological reaction
- keep the tissue moist with saline to prevent drying artifacts
*Formalin: min 8 hrs, max 72 hrs
*if >72 hrs = overhardens, autolysis
- can be cut down w/ heat, vacuum, agitation or microwave
g. Osmolality
- Hypertonic =cell shrinkage.
- Isotonic and Hypotonic = swelling and poor fixation
- best:
slightly hypertonic solutions (400-450 mOsm)
isotonic (340 mOsm)
*General principles in choice of fixative:
nature of the fixatives
type of tissue
histologic details to be demonstrated
* Practical Considerations of Fixation
1. Speed the specimen should be placed in a fixative solution
as soon as it is removed from the body to prevent autolysis and
putrefaction.
2. Penetration - Formalin diffuses into the tissue at 1 mm/hr
and slows down as it goes deeper in the tissue.
3. Volume
- traditionally, fixative used is 10-25x the tissue volume
- maximum effectiveness = 20x the tissue volume
4. Duration of Fixation Fibrous organs (uterus, intestinal tract)
take longer than small or loosely textured tissues (biopsies,
scrapings)
5. The tissue selected for sectioning should be thin enough to
allow penetration by fixative w/in a reasonable amount of time.
6. Max of 2 days, clinicians need prompt diagnosis
7.To maintain an adequate fixation time of 4-6 hours, the tissue
size must be 2cm square.
*Refrigeration is used to slow down decomposition if the tissue
that needs to be photographed and cannot be fixed
immediately
*Theres no perfect fixative, but formaldehyde is the closest
*Types Of Fixatives
1. Aldehydes
- cross-linkages formed in the proteins (b/w lysine
residues)
a. Formaldehyde
- good for immunoperoxidase techniques bec.
antigenicity is not lost
- penetrates tissue well, but is relatively slow.
- standard solution = 10% neutral buffered formalin
- buffer prevents acidity that would promote autolysis
& cause precipitation of formol-heme pigment
b. Glutaraldehyde
- fixes very quickly
- good for electron microscopy.
- penetrates very poorly
- gives best overall cytoplasmic and nuclear detail
- causes deformation of alpha-helix structure in proteins
so is not good for immunoperoxidase staining.
- Std. soln = 2% buffered glutaraldehyde
 2. Mercurials
- unknown mechanism
- contain mercury; must be disposed of carefully
- penetrate poorly and cause tissue hardness
- fast and give excellent nuclear detail
- best for hematopoietic and reticuloendothelial tissues
- mordant
- ex. mercuric chloride, B-5, Zenker's
3. Alcohols
- protein denaturants
- not used routinely bec. too much brittleness &
hardness
- very good for cytologic smears bec. they act quickly
and give good nuclear detail
- Spray cans of alcohol fixatives/ cheap hairsprays - for
PAP smears
- ex. methyl alcohol; ethyl alcohol
4. Oxidizing agents
- cross-link proteins, but cause extensive denaturation
- have specialized applications, but used very
infrequently
- permanganate fixatives (potassium permanganate)
- dichromate fixatives (potassium dichromate)
- osmium tetroxide
5. Picrates
- unknown mech of action
- with picric acid - explosion hazard in dry form
- almost as well as mercurials with nuclear detail but
does not cause as much hardness
- stains everything it touches yellow, including skin
- ex. Bouin's solution
*General Usage of Fixatives:
A. Formalin
- for all routine surgical pathology & autopsy tissues for H & E
stained slides
- most forgiving of all fixatives when conditions are not ideal
- no tissue that it will harm significantly
B. Zenker's fixatives
- for reticuloendothelial tissues; lymph nodes, spleen,
thymus, bone marrow.
- fixes nuclei very well and gives good detail.
- Caveat: the mercury deposits must be removed
(dezenkerized with iodine in xylene) before staining or black
deposits will result
C. Bouin's solution
- 750 ml Picric acid, saturated aqueous solution + 50 ml
37-40% formalin + 50 ml Glacial acetic acid
- for embryonic studies and skin
- also for testis, GI tract, endocrine, hematopoietic tissues
- excellent preservation of nuclei and chromosomes
- Long fixation: tissues become brittle and difficult to section.
*Change to 70% ethanol (less than 24 hours is optimal).
- does not require dezenkerizing before staining
D. Glutaraldehyde
- for electron microscopy
- must be cold, buffered, not more than 3 months old
- tissue must be as fresh as possible and preferably sectioned
at a thickness of no more than 1 mm to enhance fixation
E. 95% Ethanol
- for cytologic smears
- fast and cheap
*Since smears are only a cell or so thick, there is no great
problem from shrinkage
*Since smears are not sectioned, there is no problem from
induced brittleness
*Tissue Processing
- tissues made into thin microscopic sections.
- getting fixed tissue into paraffin (similar in density to tissue)
- can be sectioned from 3 - 10 microns; routine: 6-8
- main steps: dehydration & clearing
III. DEHYDRATION
- Wet fixed tissues (in aqueous solutions) cannot be directly
infiltrated with paraffin; Water is removed from tissues first
most critical stage of processing; difficult to correct
- Usually done with a series of alcohols (70% to 95% to 100%)
- Sometimes the first step is a mixture of formalin and alcohol
- Acetone is very fast, but a fire hazard, corrosive on rubber, so is
safe only for small, hand-processed sets of tissues.
- Dioxane can be used without clearing, but has toxic fumes
IV. CLEARING
- removal of the dehydrant (dealcoholization) with a substance
miscible with the embedding medium (paraffin)
-preparation for embedding
-makes tissues transparents by altering refractive index
a. Xylene most common but dangerous
b. Toluene - works well, more tolerant of small amounts of
water left in the tissues, but is 3x more expensive than xylene;
toxic
c. Chloroform used to be popular, but is a health hazard; slow
d. Methyl salicylate/ oil of wintergreen - rarely used bec.
expensive, but it smells nice
e. Benzene causes aplastic anemia
*Xylene-free substitues:
- Many are based on limolene (volatile oil in citrus peels)
- Others use long chain aliphatic hydrocarbons (Clearite); less of
a health hazard, but less forgiving with poorly fixed, dehydrated,
or sectioned tissues
-Cedarwood, aniline, clover natural oils, excellent but
slow-acting & expensive
V. INFILTRATION
- tissue is infiltrated with paraffin

VI. EMBEDDING
- must manually pick the tissues out of the cassette and pour
molten paraffin over them
- tissues are aligned or oriented properly in the block of paraffin
*vacuum can be applied inside the tissue processor to assist
penetration of the embedding agent
*Wax to be used must contain no trace of clearing agent, dust
particles, and must be rapidly cooled to reduce the wax crystal
size
1. transfer tissue w/ warm forceps to a small container of
freshly melted paraffin (tips of forceps are heated in an
alcohol lamp/forceps warmer; should be hot enough so
paraffin doesnt solidify, but not so hot as to cause paraffin
to smoke)
2. fill the bottom of the mold with a small amount of paraffin.
The depth of the mold should be at least twice the
thickness of the tissue.
3. pick up tissue & place into the mold. Manipulation must be
quick, so paraffin doesnt begin to harden.
4. Fill mold entirely with the paraffin. As the paraffin begins to
harden insert a code number label; the label should not go
down to the bottom of the paraffin.
5. Allow the surface of the paraffin block to harden, then
immerse the mold into a shallow, cool (10C) water bath for
about 10-15 min to hasten solidification of the paraffin &
prevent cracking of tissue block
6. When paraffin is completely hardened, remove it from the
mold.
*Paraffin is properly cooled & best for sectioning:
- crystals of paraffin are small and contiguous w/ each other
- clear and homogeneous
- no layering
* Embedding Medium
A. Paraffin
- melting point: 56-58 C; solid to liquid (reversible)
-maintenance temp: 2-5C above melting pt; 60-65C
- general embedding medium; 4-6 microns
*Advantages:
short time for small pieces of tissue
Thin sections can be cut with rotary microtome;
sections will adhere to each other to form a ribbon
Tissue can be stored in a dry condition indefinitely
without damage to the tissue
*Disadvantages:
Distortion, shrinkage may occur, esp when
sections are being attached to glass slides (paraffin
artifact)
Sectioning of is difficult at high temperatures
Excessive time for infiltration of large blocks
* Paraffin + dimethylsulfoxide (DMSO) elastic & resilient
Paraplast w/ plasticizers (make blocks easier to cut)
Fibrowax, Embeddol, Bioloid, Waxaco mp: 56-58C
Esterwax - mp: 46-48 C less heat, less distortion/
burning
B. Celloidin
- amorphous, slightly yellowish substance
- purified form of collodion or nitro-cellulose
-for hard tissue specimens
*Advantages:
does not require heat
has a rubbery consistency
minimal distortion of specimen
*Disadvantages:
difficult to cut thin sections
serial sections are difficult to prepare
slow process
blocks and sections must be stored in 70% alcohol
otherwise they become discolored, dry, and
shrunken
C. Low Viscosity Nitrocellulose (LVN)
*Advantages:
Low viscosity; allows higher concentration
Greater speed of impregnation
Harder, allowing thinner sections to be cut
Has a greater water tolerance than celloidin
*Disadvantages:
tendency to crack down during handling & staining
(use 0.5% oleum ricini (castor oil) to minimize this)
highly explosive
D. Double Embedding
- tissue is first impregnated with celloidin, and
subsequently blocked in paraffin wax
- used in dealing with hard tissues
- for maintenance of the morphological appearance
of the tissue
- serial sections are easily prepared
- extra degree of resilience is given when cutting
hard tissues
E. Agar
- main use is in double embedding technique with
ester wax or paraffin wax
- cohesive agent for multiple fragments or friable
tissue
F. Gelatin
- has a lower melting point than agar
- main use for whole organ sections
- for friable tissues
G. Water-soluble waxes
- Tissue can be embedded directly from water
- However, it is restricted, due to the violent
diffusion currents, which can lead to the complete
fragmentation of the section
 thin disposable variety
- advantageous for a block hidden a metal wire/ suture

H. Plastic Embedding Medium *Special Knives for Plastic blocks (methacrylate, araldite, epon)
- require special reagents for dehydration & clearing Glass knife - can section down to about 1 micron.
(expensive) Diamond knife - thin sections for electron microscopy
- few; usually done by hand (1/4 micron)
- needs Ultrathin microtome: 2-4 um
- must be small blocks for small biopsies (bone marrow/ * Types of Adhesives
liver, renal) 1. Albumin
- Background staining may be detected due to its
a. Methyl methacrylate uptake of dyes.
- very hard; good for undecalcified bone - preservative is added to prevent putrefaction
b. Glycol methacrylate (decomposition of proteins)
- most widespread; easiest to work with - Glycerol is also added to increase viscosity and
c. Araldite prevent complete drying.
- same as methacrylate; more complex - more effective if drying of sections takes place above
d. Methacrylate plastic resin Epon the coagulation point of the albumen
- routine or electron microscopy (very thin sections) - Sources: Egg albumin, Bovine, Human albumin
& immunohistochemistry
2. Gelatin
VII. BLOCKING Provides firmer attachment then albumin
- has to be gently heated before use to melt the
VIII. TRIMMING gelatin.
- Expose the sx to cutting surface - Shouldnt be kept molten for long periods as it will
- Remove excess wax, to fit into holder lose its ability to solidify.
- Truncated pyramid for appropriate sections shape & size;
parallel sides in all corners 3. Starch
Greater adhesion than gelatin
- Disadvantage: Stains with many dyes.
IX. SECTIONING
- cut into sections (thin slices of tissue: 4 15) that can be 4. Cellulose
placed on a slide in the form of 1% Methyl cellulose
*Microtome - machine on which tissue sections are cut; a knife - Advantage: Not staining to any appreciable extent
with a mechanism for advancing a paraffin block standard with commonly used in stains of histochemical rgts.
distances across it
- needs a very sharp knife! 5. Poly L-lysine
- most paraffin embedded tissues: 6 - 8 microns Use as a general-purpose section adhesive.
- No production of background staining.
*Bevel Angle (27 - 32)
- Angle b/w the cutting edge of the microtome knife 6. Sodium Silicate
*Clearance Angle (5 - 10) commercial syrup = 1:10 dilution
- b/w the surface of the block and cutting edge of the knife -Had strong adhesive properties
-= thinner sections -Advantages: Little tendency to staining with most
-= thicker sections dyes; not affected by the use of mild alkaline solutions
-Disadvantages: Blackening in some silver impregnation
*Honing techniques, in some reticulin methods, and red staining
sharpening a knife by grinding cutting edge, either on a in methyl green pyronin technique
stone or with an abrasive compound
- Heel-to-toe direction 7. Resins
- removes nicks or burrs Greatest adhesion
*Stropping - Araldite - made of epoxy resins
polishing the cutting edge of the knife on leather or canvas - Diluted 1:10 with acetone
done after honing - Little affected by most fluids in any treatment of sections
- Toe-to-heel direction
*Sectioning Artifacts
*Microtome Knife Tearing
standard thick metal variety Ripping
- allows custom sharpening to one's own satisfaction
 Venetian blinds"
Holes
Folding
*Flotation Water Bath
- helps remove wrinkles.
- sections are picked up on a glass microscopic slide
*Fishing-out Sections
1. Place the ribbon of sections into the water bath with
black base for several minutes
2. Separate the ribbon of sections into individual sections
using forceps
3. Start fishing out each section using a slide (lightly
smeared with adhesive)
4. Immerse the slide vertically
5. Fish-out a section
6. Gently lift the slide vertically
7. dry the section using a hot plate
*Oven
-15 minutes
- to help the section adhere to the slide.
- If this heat might harm such things as antigens for
immunostaining, then this step can be bypassed and
glue-coated slides used instead to pick up the sections
*Deparaffinization
- by running them through xylenes to alcohols to water.
- before any staining can be done; no stains can be done on
tissues containing paraffin
X. STAINING
- applying dyes on the sections to study architectural pattern of
the tissue and physical characteristics of the cells
- embedding process must be reversed to get the paraffin wax
out of the tissue and allow water soluble dyes to penetrate
- makes use of a variety of dyes that can stain various cellular
components of tissue
* Regressive stain
- the slides are left in the solution for a set period of time
(overstained)
- taken back through a solution such as acid-alcohol that
removes part of the stain; excess stain is removed or
decolorized from unwanted parts of the tissue and until
the desired color is obtained
- best for large batches of slides to be stained and is more
predictable on a day to day basis.
* Progressive stain
- tissue elements are stained in definite sequence with
specific periods of time
- not washed or decolorized
-the distinction of tissue detail relies solely on the
selective affinity of the dye for various cellular elements
- the slide is dipped in the hematoxylin until the desired
intensity of staining is achieved (ex. frozen section)
- simple for a single slide
- poor for batch processing
*Major groups of tissue staining
1. HISTOLOGICAL STAINING
- the tissue constituents are demonstrated in sections by
direct interaction with a dye or staining solution
(e.g. micro-anatomical stains, bacterial stains, specific
tissue stains- muscles, connective tissue and neurologic
stains)
2. HISTOCHEMICAL STAINING (HISTOCHEMISTRY)
- various constituents of tissues are studied thru chemical
reactions that permits microscopic localization of specific
tissue substances.
Perls prussian blue reaction for hemoglobin
Periodic Acid Schiff staining for carbohydrates
Enzyme histochemistry
o Active reagent - substrate
o Tissue enzymes
o The final opacity or coloration is produced
from the substrate rather than the tissue
3. IMMUNOHISTOCHEMICAL STAINING
- a combination of immunologic and histochemical
techniques that allow phenotypic markers to be detected by
antibodies (polyclonal, monoclonal, enzyme-labeled or
flourescent-labeled)
*HEMATOXYLIN & EOSIN (H & E) STAINING
- routine
- common method utilized for microanatomical studies of
tissues
-using the regressive staining which consists of :
a. overstating the nuclei
b. removal of superfluos and excessive color of the
tissue constituent by acid differentiation.
Hematoxylin
oxidized/ripened product of the logwood tree
(hematein)
naturally by putting the hematein solution on
the shelf and waiting several months
buying commercially ripened hematoxylin
putting ripening agents in the hematein
solution
- will not directly stain tissues
- needs a "mordant" or link to the tissues (metal cations:
iron, aluminum, or tungsten)
- a basic dye, affinity for the nucleic acids of the nucleus
Eosin
- acidic dye; affinity for cytoplasmic components
- much more forgiving than hematoxylin
- less of a problem in the lab.
*Steps:
1. 1st xylene bath: 3 mins
2. 2nd xylene bath: 2 to 3 mins
 3. first bath of absolute ethyl alcohol: 2 mins
4. 95% ethyl alcohol: 1 to 2 mins
5. Rinse in running water: 1 min
6. Harris Alum Hematoxylin: 5 minutes
or Ehrlichs hematoxylin: 15-30 minutes
7. Wash in running tap water to remove excess stain
8. Differentiate in 1% acid-alcohol (1mL conc. HCl to 99 mL
of 80% ethyl alcohol): 10-30 secs.
9. Rinse in tap water
10. Ammonia water: 5 minutes or 1% aqueous lithium
carbonate until the section appears blue: 30 seconds)
11. Wash in running water: 5 mins
12. Counterstain with 5% aqueous eosin: 5 minutes
or alcoholic eosin: 30 seconds or 1 minute
13. If aqueous eosin is used, wash and differentiate in tap
water under microscopic control until the nuclei appear
sharp blue to blue black and the rest is in shades of pink. If
alcoholic solution is used, differentiate with 70% alcohol.
14. Dehydrate, clear and mount
*Results:
Nuclei blue to blue black
Karyosome- dark blue
Cytoplasm- pale pink
RBCs, eosinophilic granules, keratin bright-orange red
Calcium and decalcified bone purplish blue
Decalcified bone matrix, collage and osteoid- pink
Muscle fibers- deep pink
*Overstaining can be a problem esp. w/ decalcified tissues
*DIFFERENTIATION / DECOLORIZATION
- usually done by washing the section in simple solution
(e.g. water or alcohol)
- use of acids and oxidizing agents
Primary stain basic dye
Differentation acidic solution
Further diff. - alkaline medium
*Mordant (e.g. iron alum)
can oxidize hematoxylin to a soluble, colorless compound.
- Disadvantage: a mordant stained section allowed to remain in
a differentiating agent such as 1% or 2% alcohol allows removal
of the entire dye
*METACHROMATIC STAINING
- entails the use of specific dyes which differentiate particular
substance by staining them with a color that is different from
that of the stain itself (metachromasia)
- usually employed in staining cartilage, connective tissue,
epithelial mucins, amyloid and mast cell granules
*Metachromatic dyes (basic) belongs to thizine and
triphenylmethane groups
1. Methyl violet or crystal violet
2. Cresyl blue (for reticulocytes)
3. Safranin
4. Bismarck brown
5. Basic fuchsin
6. Methylene blue
7. Thionine
8. Toluidine blue
9. Azure A, B, C
*Water- necessary for most metachromatic staining
techniques
*metachromasia is usually lost if section is dehydrated in
alcohol after staining
*Metachromasia is satisfactorily seen in formalin-fixed
tissues
*Other types of staining:
1. Direct Staining
- giving color to the sections by using alcoholic dye
solutions
-ex. methylene blue, eosin
2. Indirect Staining
process whereby the action of the dye is intensified
by adding another reagent or mordant.
- Mordant combines with a dye to to form a colored
lake which in turn combines with a tissue to form
tissue-mordant-dye-complex. The complex is
insoluble in ordinary aqueous and alcoholic solvents.
- Accentuator hastens speed of staining reaction
KOH in Loefflers MB
phenol in carbol
thionine in carbol fuchsin
3. Counterstaining
application of a different color or stain to provide
contrast and background to the staining of the
structural components to be demonstrated
4. Metallic Impregnation
specific tissue elements are demonstrated not by
stains but by colorless solutions of metallic salts which
are deposited on the surface of the tissue
5. Vital Staining
a. Intravital staining
by injecting the dye into any part of the animal
body
- ex. lithium, carmine and India ink
b. Supravital staining
used immediately after removal of cells from the
living body
- ex. neutral red, Janus green (mitochondria), Trypan
blue, Nile blue, Thionine and Toluidine Blue
*CONNECTIVE TISSUE STAINING
o Hematoxylin-eosin: Not visible bec of delicate fine
branching fibers
o Van Giesons stain: Unstained or faint pinkish color
o Periodic Acid Schiff (PAS): purplish red; not satisfactory
stain due to the delicate nature of the reticulin fibers
A. Reticulin (Reticulum) Connective Tissue Fibers - If it is desired to bring out collagen fibers sharply,
- fibrillary extracellular matrix omit the staining with acid fuchsin
a. Silver impregnation technique g. Azocarmine Stain
- Best technique because reticulin fibers are - Heidenhains modification of Malloys aniline blue
argyrophylic stain
- Prepared by producing a precipitate from silver - Valuable stain showing minute details
nitrate with sodium, potassium, or ammonium - Amyloid CT and mucuos colloid: deep blue stain
hydroxide or with lithium or sodium carbonate - Nuclei: red
- Toning with yellow gold chloride gives a very pale
gray background which improves subsequent C. Elastic Fibers
counterstaining - in skin, ligaments, aorta, arterial elastic lamina, and lung:
b. Gomoris Silver Impregnation Stain for Reticulin a. Gomoris Aldehyde-Fuchsin stain
- Ammoniacal silver solutions must be fresh
- Glassware should be washed with nitric acid and b. Weigerts Elastic Tissue stain
rinsed in distilled water - Weigerts stain: fuchsin, resorcin and ferric
- Forceps should be nonmetallic chloride
- Use glass-distilled water - diff. with acid-alcohol
- Mount the paraffin sections well - counterstained with neutral red, H & E, or
- Inactivate any unused solutions by adding excess hematoxylin and van Giesons stain
of sodium chloride solutions or of dilute - Fixation: formalin or alcohol
hydrochloric acid - Sections: thin paraffin sectios
- Elastic fibers appear dark-blue or blue-black on
B. Collagen clear background
- Forms a coarser extracellular framework than reticulin c. Verhoeffs elastic method
a. Krajjans Aniline Blue stain - Fixation: formalin
b. Silver impregnation - Section: paraffin
- Stain yellow, lavender or brown - Elastic fiber: black
c. Acid aniline dyes
- (aniline blue, acid fuchsin, methyl blue or indigo d. Krajjans Technique
carmine) from fairly strong acid solutions - Rapid method
- Fibers stain selectively - Elastic fibers: bright red
- Most commonly used acid is picric acid - Fibrin and CT: dark blue
d. Van Giesons stain - RBC: orange-yellow
- Stain red
- Simplest method using picric acid and acid fuchsin e. Orcein (Taenzer-Unna Orcein Method)
- Nuclei: brownish black to black - Vegetable dye
- Collagen (fibrous CT): pink, or deep red - Demonstrate finest and most delicate fibers in skin
- Muscle, cytoplasm, RBC, fibrin: yellow - Diff. With acid-alcohol
e. Massons Trichrome Stain - counterstained with methylene blue or alum
- Uses dyes in acid solution involving nuclear hematoxylin
staining with iron hematoxylin - Elastic fibers stain dark-brown
- cytoplasmic staining w/ a red dye (ex Ponceau
phosphotungstic acid, phosphomolydicacid or D. Fibrin
both) - insoluble fibrillar protein
- fixed staining of fibers with a blue or green stain - forming bundles which contact into dense homogenous
(e.g. aniline blue or light green) masses
- Fixation: Zenker, Helly, Boulins and formol - Seen after tissue damage, blood clots, acute inflammatory
sublimate solutions reactions
- Sections: use paraffin sections MSB Technique (Lendrums Martius, Scarlet, Blue)
- Muscle, RBC, and keratin: red - -employs Martius yellow, Brilliant crystal scarlet,
- Nuclei: blue-black and Soluble blue
- Collagen and mucus: blue - Fibrin stained red
f. Mallorys Aniline Blue Stain - early fibrin may stain yellow
- Not absolutely differential because it also stains - very old fibrin may stain blue
hyalin fibril, fibroglia fibrils, smooth and striated
muscle fibers and amyloid
- Collagen fibers: red XI. MOUNTING
- Elastic fibers: pale pink or yellow *Coverslipping
- covering with a thin piece plastic or glass:
to protect the tissue from being scratched 4. Electrolysis
to provide better optical quality for viewing under - experimental; least tissue damage
the microscope - slow and not suited for routine daily use
to preserve the tissue section for many years
- stained slide is taken through a series of alcohol solutions to *Tests for Completeness of Decalcification:
remove the water, then through clearing agents to a point at Physical / Mechanical
which a permanent resinous substance beneath the glass X ray / Radiological
coverslip, or a plastic film, can be placed over the section. Chemical
litmus paper red if due to acidity, add NH3 drop by
XII. LABELING drop blue litumus
____________________________________________________ - if cloudy: still w/ calcium
*AUTOTECHNICON - if clear: + ammonium oxalate, 30 mins cloudy if
- Automation consists of an instrument that moves the tissues incomplete
around through the various agents on a preset time scale
- most common and most reliable *Tissue softeners
- a mechanical processor with an electric motor that drives gears Perenyis: 12 24 hours
and cams 4% aqueous phenol: 1 3 days
- 1st jar: formalin (fixation) Molliflex (swollen & soapy appearance)
-last jar: paraffin (infiltration) 2 % HCl
- Newer processors have computers to control them and have 1 % HCl in 70 % alcohol
sealed reagent wells to which a vacuum and/or heat can be
applied *Post decalcification
Remove acid by saturated lithium carbonate solution
*Coldplate solidifies the paraffin wax or 5-10 % aqueous NaHCO3 for several hours
___________________________________________ Running tap water
DECALCIFICATION If EDTA is used use 70 % alcohol
- calcium deposits are extremely firm and will not section
properly with paraffin embedding owing to the difference in *Rate of decalcification
densities between calcium and paraffin (Bone specimens) More concentrated acid solutions more rapid but
- Tissue calcium must be removed prior to embedding to allow more harmful to tissue
sectioning Heat hastens decalcification, but increases damaging
- Bones, teeth, calcified tissues tuberculous lungs, effect of acids to tissues
arteriosclerotic vessels Mechanical agitation, sonication
- Poor cutting of hard tissues / knife damage Ideal time: 24 to 48 hours
- grating sensation in cutting: place block in 10% HCl (1 hr) ____________________________________________________
- Know patients case - if too large use saw ARTIFACTS IN HISTOLOGIC SECTIONS
- Change decalcifying agent regularly improper fixation
the type of fixative used
*Decalcifiers: poor dehydration and paraffin infiltration
1. Strong mineral acids improper reagents
- ex. nitric and hydrochloric acids poor microtome sectioning
- are used with dense cortical bone
- remove large quantities of calcium at a rapid rate a. Formalin-heme pigment
- can also damage cellular morphology - fine black precipitate on the slides, with no relationship to
- not recommended for delicate tissues such as bone the tissue
marrow. - can be confirmed by polarized light microscopy, because
this pigment will polarize a bright white (and the slide will
2. Organic acids look like many stars in the sky)
- ex. acetic acid; formic acid (at 10%, best all-around - forms when the formalin buffer is exhausted and the
decalcifier) tissue becomes acidic
- are better suited to bone marrow, not as harsh - most often seen in very cellular/bloody tissues, or autopsy
- act more slowly on dense cortical bone - spleen and lymph node are prone to this artifact
- Making thin sections and using enough neutral-buffered
3. EDTA formalin (10:1) will help
- can remove calcium and is not harsh (not an acid) - If the fixative solution is grossly murky brown to red, place
- penetrates tissue poorly and works slowly the tissues in new fixative.
- is expensive in large amounts
b. Large irregular clumps of black precipitate
- if fixed in a mercurial fixative such as B-5
- the tissues were not "dezenkerized" prior to staining
- will also appear white with polarized light microscopy
c. Tearing, Holes
- tissues insufficiently dehydrated prior to clearing and
infiltration with paraffin wax will be hard to section
- Tissue processor cycles should allow sufficient time for
dehydration, and final ethanol dehydrant solution should
be at 100% concentration (difficult in humid climates)
- Covering or sealing the solutions from ambient air helps
- Air conditioning (with refrigerants, not with evaporative
coolers) will also reduce humidity in the laboratory
d. Chattering, Venetian Blind appearance
- alcohols (ethanol) make excellent fixatives but tend to
make tissue sections brittle
e. Bubbles under the coverslip
- may form when the mounting media is too thin, and as it
dries, air is sucked in under the coverslip
- Contamination of clearing agents or coverslipping media
____________________________________________________
PROBLEMS IN TISSUE PROCESSING
"Floaters"
o small pieces of tissue that appear on a slide that do not
belong there (they have floated in during processing)
o may arise from sloppy procedure on the cutting bench (dirty
towels, instruments, or gloves can have tissue that is carried
over to the next case)
o do only one specimen at a time and clean thoroughly before
opening the container of the next case.
o always separate like specimens in the numbering sequence
o accession with totally different specimens so if numbers are
transposed/labels written wrong/tissue carried over, then
you will have an obvious mismatch
o If reusable cassettes are employed, tissue may potentially
be carried over and appear as "floaters" even several days
later
o The problem arises when, during embedding, not all the
tissue is removed from the cassette or not all of the wax is
removed.
o looks well-preserved--it should, because it was processed to
paraffin.
_____________________________________________________
REMINDERS
o Always be sure that you properly identify the tissue.
o The patient label on the specimen container matches that of
the request slip
o An accession number is given to the specimen
o This number must appear with the tissue at all times
o You must never submit a cassette of tissue without a label
or with the wrong label
_____________________________________________________
LABORATORY PRECAUTIONS
Bouin's solution
- made with picric acid
- sold in the aqueous state
- becomes explosive when it dries out
Sodium azide
- a preservative
- should be flushed down the drain with lots of water
to prevent the formation of metal azides in the
plumbing which is explosive
Benzidine, benzene, anthracene, napthol
- carcinogens; should not be used
Mercury-containing solutions (Zenker's or B-5)
- should always be discarded into proper containers
- If poured down a drain, will form amalgams with
the metal that build up and cannot be removed
____________________________________________________
EXAMINATION OF FRESH TISSUES
Teasing or Dissociation
Squash Preparation (Crushing)
Smear Preparation (Streaking, Spreading, Pull Apart,
Touch or Impression Smear)
Frozen Section
____________________________________________________
FROZEN SECTION
- rapid diagnosis (guide for intra-operative patient
management)
- to optimally process tissues for special studies for
diagnosis, treatment, or research
- to confirm that lesional tissue is present for diagnosis on
permanent sections (sample adequacy)
*FS Limitations:
- Limited section sampling
- Ice crystal or freezing artifact
- Inferior quality compared to paraffin sections
- Lack of special studies (time constraint)
- Special stains, immunohistochemistry, culture
- Lack of consultation for difficult cases
*Consider during RFS:
- Relevant clinical information/history
- Type of tissue or location of biopsy
- To determine beforehand what information the
surgeon requires from the FS and how the information
will be used
- Optimal turn-around time is </= 15 mins
- Coordination between lab and OR (personnel involved)
- Check cryostat (-17C)
- No fixative used
- Protection of laboratory personnel
- Selecting part of the tissue for FS
___________________________________________
MICROWAVE TECHNIQUE
- Physical agent like vacuum, oven (heat) and agitation to
increase movement of molecules and accelerate fixation
- Accelerates staining, decalcification, immunohistochemistry
and electron microscopy
- Oscillation frequency 2450 mHz
* Advantages:
 Tissue is heated right through the block in a very short
time (main advantage)
Non chemical technique (less interference)
Rapid
Lesser time for immunohistochemistry and in-situ
hybridization

*Disadvantages:
Penetrates 10-15 mm only
No significant cross linking of protein molecules;
subsequent chemical fixation may be needed
Viable spores/pathogens (alcohol based fixatives or
microwaving alone)
____________________________________________________
SPECIAL TISSUE PROCESSING

*Tissues that must be submitted unfixed

Tissues for frozen section evaluation
Gout: uric acid dissolves in formalin may use 100%
ethanol instead
Tissues submitted for infectious disease and
cytogenetic studies
Lymph nodes for lymphoma work-up
Muscle and nerve biopsy
Kidney biopsies
Tissue submitted for analysis of lipids

*Bone marrow biopsies

- Submit entire needle biopsy after fixation in Bouins
fluid overnight, w/c is mildly acidic & removes calcium.
- Cut sections at 4 microns
o Slides 1 & 5: stain with H&E
o Slides 2 & 6: reticulin stain
o Slides 3 & 7: iron
o Slides 4 & 8: store